Previously, viable chimeric flaviviruses were constructed that contained tick-borne encephalitis virus (TBEV) structural protein CME or ME genes with the remaining genes derived from dengue type 4 virus (DEN4). The ME chimera retained the neurovirulence for mice of its TBEV parent from which its M and E genes were derived, but it lacked the peripheral invasiveness of TBEV. The ME chimera was subjected to mutational analysis in an attempt to reduce or ablate neurovirulence manifest when virus is inoculated directly into the brain. Three distinct mutations were independently associated with marked reduction of mouse neurovirulence. These mutations ablated: (i) the TBEV PreM cleavage site which is required for proper processing of M protein; (ii) the TBEV E (envelope glycoprotein) glycosylation site; or (iii) the first DEN4 NS1 (non-structural protein one) glycosylation site. Each of the 3 attenuated mutants was restricted in growth in both simian and mosquito cells. Significantly, parenteral inoculation with these attenuated mutants induced complete resistance in mice to fatal encephalitis caused by subsequent challenge with the highly neurovirulent ME chimera. These observations suggest a new strategy for developing a live attenuated TBEV vaccine. Unlike the highly virulent TBEV, the naturrally occurring related Langat virus (LGT) is markedly less pathogenic for mice. Genetic analysis of the LGT may allow us to identify the molecular basis for neuroinvasiveness and neurovirulence of TBE viruses. The RNA genome of LGT (strain TP21) is 10940 nt in length and contains an open reading frame for a polyprotein of 3,414 amino acids. The 5' noncoding region is 129 nt in length of which nts. 1-25 and nts. 80-128 are conserved in the corresponding regions of the related TBEV or Powassan virus (a TBE virus of North America). LGT contains 583 nt in the 3' noncoding region of which the last 90 nt are conserved among viruses of the TBE complex. It may be possible to develop a safe and effective live attenuated TBEV vaccine by constructing DEN4-LGT chimeric viruses that express LGT antigenicity. Attenuating mutations similar to those identified earlier will be introduced in the chimeric virus genome and progeny virus analyzed for immunogenicity and loss of virulence.